GDF5 as a rejuvenating treatment for age-related neuromuscular failure.

Sarcopenia involves a progressive loss of skeletal muscle force, quality and mass during ageing, which results in increased inability and death; however, no cure has been established thus far. Growth differentiation factor 5 (GDF5) has been described to modulate muscle mass maintenance in various contexts. For our proof of concept, we overexpressed GDF5 by AAV vector injection in Tibialis Anterior (TA) muscle of adult aged (20 months) mice and performed molecular and functional analysis of skeletal muscle. We analysed human Vastus Lateralis muscle biopsies from adult young (21-42 years) and aged (77-80 years) donors, quantifying the molecular markers modified by GDF5 overexpression (OE) in mouse muscle. We validated the major effects of GDF5 overexpression using human immortalized myotubes and Schwann Cells (SCs). We established a pre-clinical study by treating chronically (for 4 months) aged mice using recombinant GDF5 protein (rGDF5) in systemic administration and evaluated the long-term effect of this treatment on muscle mass and function. Here, we demonstrated that GDF5 OE in the old TAs promoted an increase of 16.5% of muscle weight (P = 0.0471) associated with a higher percentage of 5000-6000 µm2 large fibres (P = 0.0211), without the induction of muscle regeneration. Muscle mass gain was associated with an amelioration of 26.8% of rate of force generation (P = 0.0330) and a better neuromuscular connectivity (P = 0.0098). Moreover, GDF5 OE preserved neuromuscular junction (NMJ) morphology (38.5% of nerve terminal area increase, P < 0.0001) and stimulated the expression of re-innervation-related genes, in particular markers of SCs (fold change 3.19 for S100b gene expression, P = 0.0101). To further characterize the molecular events induced by GDF5 OE during ageing, we performed a genome-wide transcriptomic analysis of treated muscles and showed that this factor leads to a "rejuvenating" transcriptomic signature in aged mice, as 42% of the transcripts dysregulated by ageing reverted to youthful expression levels upon GDF5 OE (P < 0.05). Towards a pre-clinical approach, we performed a long-term systemic treatment using rGDF5 and showed its effectiveness in counteracting age-related muscle wasting, improving muscle function (17,8% of absolute maximal force increase, P = 0.0079), ensuring neuromuscular connectivity and preventing NMJ degeneration (7,96% of AchR area increase, P = 0.0125). In addition, in human muscle biopsies, we found the same age-related alterations than those observed in mice and improved by GDF5 and reproduced its major effects on human cells, suggesting this treatment as efficient in humans. Overall, these data provide a foundation to examine the curative potential of GDF5 drug in clinical trials for sarcopenia and, eventually, other neuromuscular diseases.

Tension regulates the cartilage phenotypic expression of endplate chondrocytes through the α-catenin/actin skeleton/Hippo pathway.

The study aimed to investigate the regulatory mechanism of intracellular tension signaling in endplate chondrocytes and its impact on extracellular matrix synthesis. Human endplate chondrocytes were subjected to tension load using Flexcell FX-5000™, and changes in phenotype, morphology, and the expression of Hippo signaling pathway and α-Catenin were assessed through various techniques. Through the overexpression of YAP and inhibition of α-Catenin, the study clarified the intracellular tension signaling pathway and its regulation of extracellular matrix synthesis in endplate cartilage. In vitro-cultured human endplate chondrocytes significantly suppressed phenotype-related genes and proteins, accompanied by distinct changes in cytoskeleton morphology. Tension activation resulted in the substantial activation of the Hippo pathway, increased phosphorylation of YAP, and reduced nuclear translocation of YAP. YAP overexpression alleviated the inhibitory effect of tension on extracellular matrix synthesis in endplate chondrocytes. Tension also upregulated the expression of α-Catenin in endplate chondrocytes, which was attenuated by inhibiting α-Catenin expression, thereby reducing the impact of tension on cytoskeletal morphology and YAP nuclear translocation. Taken together, the α-Catenin/actin skeleton/Hippo-coupled network is a crucial signaling pathway for tension signaling in endplate chondrocytes, providing potential therapeutic targets for the treatment of endplate cartilage degeneration.

Postnatal Pdzrn3 deficiency causes acute muscle atrophy without alterations in endplate morphology.

PDZ domain-containing RING finger family protein 3 (PDZRN3) is expressed in various tissues, including the skeletal muscle. Although PDZRN3 plays a crucial role in the terminal differentiation of myoblasts and synaptic growth/maturation in myogenesis, the role of this molecule in postnatal muscles is completely unknown despite its lifelong expression in myofibers. In this study, we aimed to elucidate the function of PDZRN3 in mature myofibers using myofiber-specific conditional knockout mice. After tamoxifen injection, PDZRN3 deficiency was confirmed in both fast and slow myofibers of Myf6-CreERT2; Pdzrn3flox/flox (Pdzrn3mcKO) mice. Transcriptome analysis of the skeletal muscles of Pdzrn3mcKO mice identified differentially expressed genes, including muscle atrophy-related genes such as Smox, Amd1/2, and Mt1/2, suggesting that PDZRN3 is involved in the homeostatic maintenance of postnatal muscles. PDZRN3 deficiency caused muscle atrophy, predominantly in fast-twitch (type II) myofibers, and reduced muscle strength. While myofiber-specific PDZRN3 deficiency did not influence endplate morphology or expression of neuromuscular synaptic formation-related genes in postnatal muscles, indicating that the relationship between PDZRN3 and neuromuscular junctions might be limited during muscle development. Considering that the expression of Pdzrn3 in skeletal muscles was significantly lower in aged mice than in mature adult mice, we speculated that the PDZRN3-mediated muscle maintenance system might be associated with the pathophysiology of age-related muscle decline, such as sarcopenia.

Qualitative and Quantitative MR Imaging of the Cartilaginous Endplate: A Review.

The cartilaginous endplate (CEP) plays a pivotal role in facilitating the supply of nutrients and, transport of metabolic waste, as well as providing mechanical support for the intervertebral disc (IVD). Recent technological advances have led to a surge in MR imaging studies focused on the CEP. This article describes the anatomy and functions of the CEP as well as MRI techniques for both qualitative and quantitative assessment of the CEP. Effective CEP MR imaging sequences require two key features: high spatial resolution and relatively short echo time. High spatial resolution spoiled gradient echo (SPGR) and ultrashort echo time (UTE) sequences, fulfilling these requirements, are the basis for most of the sequences employed in CEP imaging. This article reviews existing sequences for qualitative CEP imaging, such as the fat-suppressed SPGR and UTE, dual-echo subtraction UTE, inversion recovery prepared and fat-suppressed UTE, and dual inversion recovery prepared UTE sequences. These sequences are employed together with other techniques for quantitative CEP imaging, including measurements of T2*, T2, T1, T1ρ, magnetization transfer, perfusion, and diffusion tensor parameters. EVIDENCE LEVEL: 1 TECHNICAL EFFICACY: Stage 2.

Quantal size increase induced by the endocannabinoid 2-arachidonoylglycerol requires activation of CGRP receptors in mouse motor synapses.

In mouse motor synapses, the exogenous application of the endocannabinoid (EC) 2-arachidonoylglycerol (2-AG) increases acetylcholine (ACh) quantal size due to the activation of CB1 receptors and the stimulation of ACh vesicular uptake. In the present study, microelectrode recordings of miniature endplate potentials (MEPP) revealed that this effect of 2-AG is independent of brain-derived neurotrophic factor (BDNF) signaling but involves the activation of calcitonin gene-related peptide (CGRP) receptors along with CB1 receptors. Potentiation of MEPP amplitude in the presence of 2-AG was prevented by blockers of CGRP receptors and ryanodine receptors (RyR) and by inhibitors of phospholipase C (PLC) and Ca2+ /calmodulin-dependent protein kinase II (CaMKII). Therefore, we suggest a hypothetical chain of events, which starts from the activation of presynaptic CB1 receptors, involves PLC, RyR, and CaMKII, and results in CGRP release with the subsequent activation of presynaptic CGRP receptors. Activation of CGRP receptors is probably a part of a complex molecular cascade leading to the 2-AG-induced increase in ACh quantal size and MEPP amplitude. We propose that the same chain of events may also take place if 2-AG is endogenously produced in mouse motor synapses, because the increase in MEPP amplitude that follows after prolonged tetanic muscle contractions (30 Hz, 2 min) was prevented by the blocking of CB1 receptors. This work may help to unveil the previously unknown aspects of the functional interaction between ECs and peptide modulators aimed at the regulation of quantal size and synaptic transmission.

BDNF/TrkB signalling, in cooperation with muscarinic signalling, retrogradely regulates PKA pathway to phosphorylate SNAP-25 and Synapsin-1 at the neuromuscular junction.

BACKGROUND: Protein kinase A (PKA) enhances neurotransmission at the neuromuscular junction (NMJ), which is retrogradely regulated by nerve-induced muscle contraction to promote Acetylcholine (ACh) release through the phosphorylation of molecules involved in synaptic vesicle exocytosis (SNAP-25 and Synapsin-1). However, the molecular mechanism of the retrograde regulation of PKA subunits and its targets by BDNF/TrkB pathway and muscarinic signalling has not been demonstrated until now. At the NMJ, retrograde control is mainly associated with BDNF/TrkB signalling as muscle contraction enhances BDNF levels and controls specific kinases involved in the neurotransmission. Neurotransmission at the NMJ is also highly modulated by muscarinic receptors M1 and M2 (mAChRs), which are related to PKA and TrkB signallings. Here, we investigated the hypothesis that TrkB, in cooperation with mAChRs, regulates the activity-dependent dynamics of PKA subunits to phosphorylate SNAP-25 and Synapsin-1. METHODS: To explore this, we stimulated the rat phrenic nerve at 1Hz (30 minutes), with or without subsequent contraction (abolished by µ-conotoxin GIIIB). Pharmacological treatments were conducted with the anti-TrkB antibody clone 47/TrkB for TrkB inhibition and exogenous h-BDNF; muscarinic inhibition with Pirenzepine-dihydrochloride and Methoctramine-tetrahydrochloride for M1 and M2 mAChRs, respectively. Diaphragm protein levels and phosphorylation' changes were detected by Western blotting. Location of the target proteins was demonstrated using immunohistochemistry. RESULTS: While TrkB does not directly impact the levels of PKA catalytic subunits Cα and Cß, it regulates PKA regulatory subunits RIα and RIIß, facilitating the phosphorylation of critical exocytotic targets such as SNAP-25 and Synapsin-1. Furthermore, the muscarinic receptors pathway maintains a delicate balance in this regulatory process. These findings explain the dynamic interplay of PKA subunits influenced by BDNF/TrkB signalling, M1 and M2 mAChRs pathways, that are differently regulated by pre- and postsynaptic activity, demonstrating the specific roles of the BDNF/TrkB and muscarinic receptors pathway in retrograde regulation. CONCLUSION: This complex molecular interplay has the relevance of interrelating two fundamental pathways in PKA-synaptic modulation: one retrograde (neurotrophic) and the other autocrine (muscarinic). This deepens the fundamental understanding of neuromuscular physiology of neurotransmission that gives plasticity to synapses and holds the potential for identifying therapeutic strategies in conditions characterized by impaired neuromuscular communication.

Research Progress on the Role of Cartilage Endplate in Intervertebral Disc Degeneration.

Low back pain significantly impacts individuals' quality of life, with intervertebral disc degeneration (IDD) being a primary contributor to this condition. Currently, IDD treatment primarily focuses on symptom management and does not achieve a definitive cure. The cartilage endplate (CEP), a crucial nutrient-supplying tissue of the intervertebral disc, plays a pivotal role in disc degeneration. This review examines the mechanisms underlying CEP degeneration, summarizing recent advancements in understanding the structure and function of CEP, the involvement of various signaling pathways, and the roles of cartilage endplate stem cells (CESCs) and exosomes (Exos) in this process. The aim of this review is to provide a comprehensive reference for future research on CEP. Despite progress in understanding the role of CEP in IDD, the mechanisms underlying CEP degeneration remain incompletely elucidated. Future research poses significant challenges, necessitating further investigations to elucidate the complexities of CEP.

Chondrocyte-targeted exosome-mediated delivery of Nrf2 alleviates cartilaginous endplate degeneration by modulating mitochondrial fission.

BACKGROUND: Cartilaginous endplate (CEP) degeneration, which is an important contributor to intervertebral disc degeneration (IVDD), is characterized by chondrocyte death. Accumulating evidence has revealed that dynamin-related protein 1 (Drp1)-mediated mitochondrial fission and dysfunction lead to apoptosis during CEP degeneration and IVDD. Exosomes are promising agents for the treatment of many diseases, including osteoporosis, osteosarcoma, osteoarthritis and IVDD. Despite their major success in drug delivery, the full potential of exosomes remains untapped. MATERIALS AND METHODS: In vitro and in vivo models of CEP degeneration were established by using lipopolysaccharide (LPS). We designed genetically engineered exosomes (CAP-Nrf2-Exos) expressing chondrocyte-affinity peptide (CAP) on the surface and carrying the antioxidant transcription factor nuclear factor E2-related factor 2 (Nrf2). The affinity between CAP-Nrf2-Exos and CEP was evaluated by in vitro internalization assays and in vivo imaging assays. qRT‒PCR, Western blotting and immunofluorescence assays were performed to examine the expression level of Nrf2 and the subcellular localization of Nrf2 and Drp1. Mitochondrial function was measured by the JC-1 probe and MitoSOX Red. Mitochondrial morphology was visualized by MitoTracker staining and transmission electron microscopy (TEM). After subendplate injection of the engineered exosomes, the degree of CEP degeneration and IVDD was validated radiologically and histologically. RESULTS: We found that the cargo delivery efficiency of exosomes after cargo packaging was increased by surface modification. CAP-Nrf2-Exos facilitated chondrocyte-targeted delivery of Nrf2 and activated the endogenous antioxidant defence system in CEP cells. The engineered exosomes inhibited Drp1 S616 phosphorylation and mitochondrial translocation, thereby preventing mitochondrial fragmentation and dysfunction. LPS-induced CEP cell apoptosis was alleviated by CAP-Nrf2-Exo treatment. In a rat model of CEP degeneration, the engineered exosomes successfully attenuated CEP degeneration and IVDD and exhibited better repair capacity than natural exosomes. CONCLUSION: Collectively, our findings showed that exosome-mediated chondrocyte-targeted delivery of Nrf2 was an effective strategy for treating CEP degeneration.

CT-based surrogate parameters for MRI-based disc height and endplate degeneration in the lumbar spine.

PURPOSE: This study investigated potential use of computed tomography (CT)-based parameters in the lumbar spine as a surrogate for magnetic resonance imaging (MRI)-based findings. METHODS: In this retrospective study, all individuals, who had a lumbar spine CT scan and MRI between 2006 and 2012 were reviewed (n = 198). Disc height (DH) and endplate degeneration (ED) were evaluated between Th12/L1-L5/S1. Statistics consisted of Spearman correlation and univariate/multivariable regression (adjusting for age and gender). RESULTS: The mean CT-DH increased kranio-caudally (8.04 millimeters (mm) at T12/L1, 9.17 mm at L1/2, 10.59 mm at L2/3, 11.34 mm at L3/4, 11.42 mm at L4/5 and 10.47 mm at L5/S1). MRI-ED was observed in 58 (29%) individuals. CT-DH and MRI-DH had strong to very strong correlations (rho 0.781-0.904, p < .001). MRI-DH showed higher absolute values than CT-DH (mean of 1.76 mm). There was a significant association between CT-DH and MRI-ED at L2/3 (p = .006), L3/4 (p = .002), L4/5 (p < .001) and L5/S1 (p < .001). A calculated cut-off point was set at 11 mm. CONCLUSIONS: In the lumbar spine, there is a correlation between disc height on CT and MRI. This can be useful in trauma and emergency cases, where CT is readily available in the lack of an MRI. In addition, in the middle and lower part of the lumbar spine, loss of disc height on CT scans is associated with more pronounced endplate degeneration on MRIs. If the disc height on CT scans is lower than 11 mm, endplate degeneration on MRIs is likely more pronounced. LEVEL AND DESIGN: Level III, a retrospective study.

Novel Modic grading scoring system and its clinical validation: a preliminary investigation.

PURPOSE: To propose a novel Modic grading scoring system and explore the relationship between the Modic grading score and disc degeneration, disc herniation, disc height, and clinical symptom scores. METHOD: In total, 194 patients were included in the study. The new Modic grading scoring system included four indicators: invaded vertebral height, invaded endplate length, endplate morphology, and grade of endplate defects. The severity of Modic changes was visually quantified by numerical scores, and the kappa value was used to verify the interobserver and intraobserver reliability. Spearman correlation analysis was used to explore the relationship between the Modic grading score and intervertebral disc degeneration, disc herniation, disc height, and clinical symptom scores. RESULTS: The interobserver and intraobserver reliability showed substantial to almost perfect agreement in the new Modic grading scoring system. The Modic grading score was positively correlated with intervertebral disc degeneration (r = 0.757, p < 0.001) and negatively correlated with the intervertebral disc height index (r = - 0.231, p < 0.001). There was no significant correlation between the Modic grading scoring system and disc herniation (r = 0.369, p = 0.249). Additionally, there was no significant correlation between the Modic grading score and the Japanese Orthopaedic Association score (r = - 0.349, p = 0.25), Oswestry Disability Index score (r = 0.246, p = 0.11), or visual analogue scale score (r = 0.315, p = 0.35). CONCLUSION: The new Modic grading scoring system had good interobserver and intraobserver reliability. The Modic grading score was positively correlated with intervertebral disc degeneration and negatively correlated with the intervertebral disc height.